Novel retinoic acid compound, pharmaceutical composition comprising same, and use thereof

Abstract

The present invention provides a compound or a pharmaceutically acceptable salt thereof, and a pharmaceutical composition thereof, wherein the compound comprises retinoic acid conjugated to a carbohydrate. In addition, the invention also provides application of the compound or the pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparation of drugs for inhibiting virus infection or replication or treating cancers.

Description

Technical Field

[0001] The present invention relates to a novel retinoic acid compound, a pharmaceutical composition containing the same and use thereof in the manufacture of a medicament for inhibiting viral infection or replication or for treating cancer. Background Art

[0002] Coronaviruses are a group of positive, single-stranded RNA viruses belonging to the Coronaviridae family, which include seven species / strains that infect humans, namely severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), and human coronavirus HKU (HCoV-HKU1). It is worth noting that SARS-CoV-2 has been identified as the strain that causes the coronavirus disease (COVID-19) pandemic.

[0003] The genomes of SARS-CoV-2 and SARS-CoV have high sequence identity. Both SARS-CoV-2 and SARS-CoV are highly dependent on the activity of two viral proteases, namely 3C-like protease (3CLpro, also known as main protease (Mpro) or non-structural protein 5 (nsp5)) and papain-like protease (PLpro, which is the protease domain of non-structural protein 3 (nsp3)), to achieve the viral proliferation cycle and viral transmission. It has been reported that all-trans retinoic acid (ATRA, also known as vitamin A acid or retinoin) can be considered as a potential therapeutic agent against SARS-CoV-2 by inhibiting the activity of 3CLpro.

[0004] In addition to 3CLpro, PLpro is also a potential target because this enzyme plays an essential role in the cleavage and maturation of viral polyproteins, the assembly of replicase-transcriptase complexes, and the disruption of host responses. Although the main function of Mpro and PLpro is to process viral polyproteins in a synergistic manner, PLpro also has the additional function of stripping ubiquitin and interferon-stimulatory gene factor 15 (ISG15) from host cell proteins to allow coronaviruses to evade the host's innate immune response. In other words, PLpro is not only related to viral replication, but also to the dysregulation of the signaling cascade in infected cells, which leads to cell death of surrounding uninfected cells. Therefore, drugs designed to inhibit PLpro function also have the potential to fight SARS-CoV-2. Recently, in addition to ATRA, some researchers have reported that its derivative 13-cis retinoic acid (also known as isotretinoin) is a potential PLpro inhibitor and can be used to treat COVID-19 caused by SARS-CoV-2.

[0005] In addition, retinoic acid (including ATRA and 13-cis retinoic acid) has become a promising compound for the treatment of various cancers due to its specific effects on cell proliferation, differentiation and apoptosis and low toxicity. Retinoic acid receptors in the human cell nucleus have been found by biochemists and found not to be mutated in cancer cells, so retinoic acid may exert its anti-cancer effects in many malignancies. For example, in children with high-risk neuroblastoma, treatment with 13-cis retinoic acid was found to reduce the risk of cancer recurrence after high-dose chemotherapy and stem cell transplantation. ATRA has been studied in combination with other drugs for various cancers and precancerous lesions. Several clinical trials are currently underway using ATRA as part of combination therapy. For example, ATRA combined with different interferons (IFNs) has been shown to enhance the effects of both drugs and lead to growth inhibition and cell death of tumor cell lines. Nevertheless, in order to unlock the therapeutic potential of retinoic acid, many studies have emphasized the need to better understand the mechanisms of blocking retinoic acid signaling and retinoic acid-regulated gene expression in cancers such as acute myeloid leukemia (AML). It is clear that combination therapy targeting multiple gene silencing mechanisms may be the most effective strategy to reactivate ATRA-sensitive gene expression and AML cell differentiation and mediate the general anticancer activity of ATRA. Currently, the identification of protein classes that control gene expression through histone and DNA modifications can promote the development of novel therapeutic agents, so-called epigenetic drugs that alter chromatin structure. However, such epigenetic modifying drugs have been shown to be only partially effective against different cancers when used alone. Summary of the invention

[0006] Based on the above reasons, the present invention provides a novel retinoic acid compound and a pharmaceutical composition comprising the same, which can effectively inhibit viral infection or replication, or treat cancer.

[0007] In one aspect of the present invention, a compound or a pharmaceutically acceptable salt thereof is provided, wherein the compound comprises retinoic acid conjugated to a carbohydrate.

[0008] Preferably, the compound is represented by formula (I):

[0009]

[0010] Where R 1 is a substituted or unsubstituted functional group of the carbohydrate.

[0011] Preferably, the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides and polysaccharides.

[0012] Preferably, the oligosaccharide is a mono-oligosaccharide or a hetero-oligosaccharide; and the polysaccharide is a mono-polysaccharide or a hetero-polysaccharide.

[0013] Preferably, the carbohydrate comprises glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3 / 1,6-glucan oligosaccharides, raffinose, stachyose, verbascose, fructooligosaccharides, starch, glycogen, cellulose or any combination thereof.

[0014] Preferably, the compound or a pharmaceutically acceptable salt thereof is encapsulated by liposomes.

[0015] In another aspect of the present invention, a pharmaceutical composition is provided, which includes the above-mentioned compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0016] Preferably, the pharmaceutically acceptable carrier comprises liposomes, and the compound or its pharmaceutically acceptable salt is encapsulated by the liposomes.

[0017] Preferably, the pharmaceutical composition further comprises metal ions.

[0018] Preferably, the metal ions include monovalent ions, divalent ions or a combination thereof. More preferably, the monovalent ions include K + 、Na + or a combination thereof; and the divalent ions include Zn 2+ Mg 2+ , Cu 2+ , Mn 2+ , Ca 2+ , Fe 2+ or any combination thereof.

[0019] Preferably, the pharmaceutically acceptable carrier comprises liposomes, and the metal ions are encapsulated by the liposomes.

[0020] Preferably, the compound or a pharmaceutically acceptable salt thereof and the metal ion are encapsulated by the liposome individually or simultaneously.

[0021] In yet another aspect, the present invention provides use of the compound or its pharmaceutically acceptable salt or the pharmaceutical composition in the manufacture of a medicament for inhibiting viral infection or replication.

[0022] In yet another aspect, the present invention provides a use of the compound or its pharmaceutically acceptable salt or the pharmaceutical composition in the manufacture of a medicament for treating cancer.

[0023] Therefore, the present invention provides at least the following advantages:

[0024] 1. Compared with retinoic acid alone, the compounds of the present invention and their pharmaceutical compositions can enhance the ability to inhibit viral infection and / or replication, wherein the compounds are retinoic acid conjugated to carbohydrates.

[0025] 2. The compounds of the present invention and their pharmaceutical compositions can be used as potential drugs against coronaviruses (especially SARS-CoV-2 that causes COVID-19).

[0026] 3. The compounds of the present invention and their pharmaceutical compositions can also enhance the ability to inhibit cancer cells, thereby effectively treating various cancers, such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer and liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The results of liquid chromatography-mass spectrometry (LC-MS) analysis of compound (Ia) according to an example of the present invention are described.

[0028] Figure 2 A graph illustrating the change in absorbance over time in a SARS-CoV-2 PLpro inhibition assay for novel retinoic acid compounds according to examples of the present invention.

[0029] Figure 3 TCID 50 , which shows the antiviral effect of the novel retinoic acid compound according to an embodiment of the present invention in the SARS-CoV-2 animal model.

[0030] Figure 4A graph illustrating cell viability shows the cytotoxic effect of novel retinoic acid compounds according to examples of the present invention on AsPC-1 cancer cell line.

[0031] Figure 5 A graph illustrating cell viability shows the cytotoxic effect of novel retinoic acid compounds according to examples of the present invention on MDA-MB-231 cancer cell lines.

[0032] Figure 6 A graph illustrating cell viability shows the cytotoxic effect of novel retinoic acid compounds according to examples of the present invention on HCT-116 cancer cell lines.

[0033] Figure 7 A graph illustrating cell viability shows the cytotoxic effect of novel retinoic acid compounds according to examples of the present invention on Huh-7 cancer cell lines.

[0034] Figure 8 A graph illustrating cell viability shows the cytotoxic effect of novel retinoic acid compounds according to examples of the present invention on SKOV-3 cancer cell lines.

[0035] Fig. 9 A graph illustrating cell viability shows the cytotoxic effect of novel retinoic acid compounds according to examples of the present invention on A549 cancer cell lines.

[0036] Fig.10 A graph illustrating cell viability shows the cytotoxic effect of novel retinoic acid compounds according to examples of the present invention on H460 cancer cell lines.

[0037] Fig.11 A graph illustrating the change in absorbance over time in a SARS-CoV-2 PLpro inhibition test of a second pharmaceutical composition according to an embodiment of the present invention.

[0038] Fig.12 TCID 50 , which shows the antiviral effect of the second pharmaceutical composition according to an embodiment of the present invention in a SARS-CoV-2 animal model. DETAILED DESCRIPTION

[0039] Although the present invention is susceptible to various modifications and alternative forms, specific embodiments are shown and described in detail by way of example in the drawings. However, it should be understood that this description is not intended to limit the present invention to specific embodiments, on the contrary, the present invention will cover all modifications, equivalents and alternative forms that fall within the spirit and scope of the present invention.

[0040] definition

[0041] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. It will be further understood that, unless explicitly defined as such herein, terms defined in commonly used dictionaries should be interpreted as having the same meaning as in the context of the relevant technology and will not be interpreted in an idealized or overly formal sense.

[0042] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the embodiments of the present invention. Unless the context clearly indicates otherwise, the singular forms "a", "an" and "said" used herein are also intended to include the plural forms. It will be further understood that the terms "comprise", "comprising", "including" and / or "comprising", when used herein, specify the presence of the described features, integers, steps, operations, components, parts and / or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, components, parts and / or combinations thereof.

[0043] The term "subject" herein means a mammal for which diagnosis, prognosis or treatment is desired. Generally, the mammal is a human. In certain embodiments, the mammal may refer to a non-human mammal, such as a non-human primate, cow, horse, goat, sheep, dog, cat, rabbit, pig, mouse or rat, used, for example, to screen, characterize and evaluate drugs and therapies.

[0044] The term "administering" or "administered" herein means introducing, providing or delivering the intended active ingredient to a subject by any applicable route to perform its intended function.

[0045] The term "cancer" as used herein refers to leukemias, lymphomas, carcinomas, sarcomas, and other malignancies that may grow indefinitely, which may spread locally by invasion and systemically by metastasis. Examples of cancer include, but are not limited to, ovarian cancer, adrenal cancer, bone cancer, brain cancer, breast cancer, bronchial cancer, colorectal cancer and / or rectal cancer, gallbladder cancer, head and neck cancer, kidney cancer, laryngeal cancer, liver cancer, lung cancer, cancer of nervous tissue, pancreatic cancer, prostate cancer, parathyroid cancer, skin cancer, stomach cancer, and thyroid cancer. Some other examples of cancer include cholangiocarcinoma, acute and chronic lymphocytic and granular cell tumors, adenocarcinoma, adenoma, basal cell carcinoma, dysplastic epithelial cells and carcinoma in situ of the cervix, Ewing's sarcoma, epidermoid carcinoma, giant cell tumor, glioblastoma multiforma, hair cell tumor, ganglioneuroma of the small intestine, hyperplastic corneal neuroma, islet cell carcinoma, Kaposi's sarcoma, leiomyoma, malignant carcinoid, malignant melanoma, malignant hypercalcemia, marfanoid habitus tumor, medullary carcinoma, metastatic skin cancer, mucosal neuroma, myeloma, mycosis fungoides, neuroblastoma, osteosarcoma, pheochromocytoma, polycythermia vera, vera), primary brain tumors, small cell lung tumors, ulcerative and papillary squamous cell carcinomas, cell hyperplasia, spermatoma, soft tissue sarcoma, retinoblastoma, rhabdomyosarcoma, renal cell tumor, localized skin lesions, reticulum cell sarcoma, and Wilm's tumor.

[0046] The term "oligosaccharide" herein means a carbohydrate composed of a few monosaccharides, usually about three to ten monosaccharide units. Among them, oligosaccharides with one type of monosaccharide subunit are called mono-oligosaccharides; oligosaccharides with more than one type of monosaccharide subunits are called hetero-oligosaccharides.

[0047] The term "polysaccharide" herein means a carbohydrate composed of a large number of monosaccharide units. Among them, polysaccharides having one monosaccharide subunit are called monopolysaccharides; polysaccharides having more than one monosaccharide subunit are called heteropolysaccharides.

[0048] The term "liposome" as used herein means a particle characterized by having an aqueous interior space separated from the external medium by a membrane forming one or more bilayers of the vesicle. The main types of liposomes are multilamellar vesicles (MLVs, lipid bilayers with several lamellar phases), small unilamellar liposome vesicles (SUVs, with a single lipid bilayer) and large unilamellar vesicles (LUVs, with a single lipid bilayer). The bilayer membranes of unilamellar or multilamellar vesicles are typically formed by lipids (i.e., amphiphilic molecules of synthetic or natural origin comprising spatially separated hydrophobic and hydrophilic domains).

[0049] Description of Embodiments

[0050] Hereinafter, exemplary embodiments of the present invention will be described in more detail with reference to the accompanying drawings.

[0051] In one embodiment, a compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the compound (hereinafter referred to as the "first" pharmaceutical composition) is provided, wherein the compound comprises retinoic acid conjugated to a carbohydrate.

[0052] In an exemplary embodiment, the compound is represented by formula (I):

[0053]

[0054] Where R 1 is a substituted or unsubstituted functional group of the carbohydrate.

[0055] In certain embodiments, the concentration of the compound comprising retinoic acid conjugated to a carbohydrate may be, but is not limited to, 0.1 μM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 500 μM, 0.1 μM to 250 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0056] In another embodiment, a pharmaceutical composition is provided, which includes retinoic acid and a carbohydrate (hereinafter referred to as the "second" pharmaceutical composition). In certain embodiments, the retinoic acid is 13-cis retinoic acid (also known as isoretinic acid).

[0057] In certain embodiments, the concentration of retinoic acid may be, but is not limited to, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0058] In certain embodiments, the carbohydrate may be a monosaccharide, a disaccharide, an oligosaccharide or a polysaccharide. The oligosaccharide may be a monooligosaccharide or a heterooligosaccharide. The polysaccharide may be a monopolysaccharide or a heteropolysaccharide. The monosaccharide may be selected from (but not limited to) glucose, fructose, galactose and mannose. The disaccharide may be selected from (but not limited to) sucrose, lactose and maltose. The oligosaccharide may be selected from (but not limited to) β-1,3 / 1,6-glucan oligosaccharide, raffinose, stachyose, verbascose and fructooligosaccharide. The polysaccharide may be selected from (but not limited to) starch, glycogen and cellulose.

[0059] In certain embodiments, the concentration of the carbohydrate may be, but is not limited to, 0.1 μM to 200 mM, 0.1 μM to 150 mM, 0.1 μM to 100 mM, 0.1 μM to 10 mM, 0.1 μM to 1 mM, 0.1 μM to 500 μM, 0.1 μM to 250 μM, 0.1 μM to 100 μM, 0.1 μM to 50 μM, 1 μM to 200 mM, 1 μM to 150 mM, 1 μM to 10 0 mM, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 200 mM, 10 μM to 150 mM, 10 μM to 100 mM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0060] In certain embodiments, the molar ratio of retinoic acid to carbohydrate in the second pharmaceutical composition may be about 1:10. -3 to 100, 1:10 -3 to 20, 1:1 to 20 or 1:1 to 10.

[0061] In certain embodiments, the first pharmaceutical composition and the second pharmaceutical composition each optionally further include a metal ion. Preferably, the metal ion includes a monovalent ion, a divalent ion, or a combination thereof. In certain embodiments, the monovalent ion includes K + 、Na + or a combination thereof; and divalent ions including Zn 2+ Mg 2+ , Cu 2+ , Mn 2+ , Ca2+ , Fe 2+ or any combination thereof.

[0062] In certain embodiments, the concentration of the metal ion may be 1 μM to 300 mM, 1 μM to 250 mM, 1 μM to 200 mM, 1 μM to 150 mM, 1 μM to 100 mM, 1 μM to 10 mM, 1 μM to 1 mM, 1 μM to 500 μM, 1 μM to 250 μM, 1 μM to 100 μM, 1 μM to 50 μM, 10 μM to 300 mM, 10 μM to 250 mM, 10 μM to 200 mM, 10 μM to 150 mM, 10 μM to 100 mM, 10 μM to 10 mM, 10 μM to 1 mM, 10 μM to 500 μM, 10 μM to 250 μM, 10 μM to 100 μM, or 10 μM to 50 μM.

[0063] In certain embodiments, the molar ratio of the compound to the metal ion in the first pharmaceutical composition may be about 1:10. -3 Up to 10 3 , 1:0.1 to 20, 1:0.1 to 10 or 1:1 to 10.

[0064] In certain embodiments, the molar ratio of retinoic acid, carbohydrate, and metal ion in the second pharmaceutical composition may be about 1:10. -4 Until 20:10 -4 Up to 10 3 , 1:10 -4 Until 20:10 -3 Up to 10 3 , 1:10 -4 Until 20:10 -3 to 20, 1:10 -4 to 20: 0.1 to 20, 1:10 -4 to 20:1 to 10, 1:0.1 to 20:10 -4 Up to 10 3 1:0.1 to 20:10 -3 Up to 10 3 1:0.1 to 20:10 -3 to 20, 1:0.1 to 20:0.1 to 20, 1:0.1 to 20:1 to 20, 1:0.1 to 20:1 to 20, 1:0.1 to 20:1 to 10, 1:0.1 to 1:10 -4 Up to 10 3 1:0.1 to 1:10 -3 Up to 10 3 1:0.1 to 1:10 -3 to 20, 1:0.1 to 1:0.1 to 20, 1:0.1 to 1:1 to 20 or 1:0.1 to 1:1 to 10.

[0065] In certain embodiments, the first pharmaceutical composition and the second pharmaceutical composition each optionally further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is widely used in the field of drug manufacturing. Examples of pharmaceutically acceptable carriers may include (but are not limited to) liposomes, excipients, adjuvants, solvents, buffers, emulsifiers, suspending agents, decomposing agents, disintegrants, dispersants, adhesives, stabilizers, chelating agents, diluents, gelling agents, preservatives, wetting agents, lubricants, absorption delay agents and the like. The selection and dosage of pharmaceutically acceptable carriers are within the professional knowledge of ordinary technicians in the field.

[0066] In certain embodiments, the pharmaceutically acceptable carrier is a liposome. The compound of the present application or its pharmaceutically acceptable salt is encapsulated by the liposome. The compound or its pharmaceutically acceptable salt and the metal ion in the first pharmaceutical composition can be encapsulated by the liposome alone or simultaneously. The retinoic acid and the carbohydrate in the second pharmaceutical composition can be encapsulated by the liposome alone or simultaneously. All or at least two of the retinoic acid, carbohydrate and metal ion in the second pharmaceutical composition can be encapsulated by the liposome alone or simultaneously.

[0067] Exemplary liposomes may be neutral, positively charged or negatively charged. In general, lipids commonly used in liposomes typically include dialiphatic chain lipids, such as phospholipids, diglycerides and diglycerol lipids; single lipids, such as sphingomyelin and glycosphingolipids; steroids, such as cholesterol and its derivatives, and combinations thereof. Examples of phospholipids include, but are not limited to, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soybean phosphatidylcholine (DPPC), and 1,2-diol-sn-glycero-3-phosphocholine (DPPC). soyphosphatidylcholine, HSPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DPPG), l-palmitoyl-2-stearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (PSPG), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt) (DSPG), 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPG), 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DPPS), 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DSPS), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dimyristoyl-sn-glycero-3-phosphate (sodium salt) (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt) (DPPA), 1,2-distearoyl-sn-glycero-3-phosphate (sodium salt) (DSPA), 1,2-Dioleoyl-sn-glycero-3-phosphate (sodium salt) (DOPA), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myo-inositol) (ammonium salt) (DPPI), 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt) (DSPI), 1 ,2-dioleoyl-sn-glycero-3-phospho-(1-myo-inositol) (ammonium salt) (DOPI), cardiolipin, L-α-phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (18:1EPC), L-α-phosphatidylethanolamine (EPE), dimethyldioctadecyl ammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol hydrochloride. ,

[0068] Suitable lipids may be lipid mixtures of one or more of the aforementioned lipids, or mixtures of one or more of the aforementioned lipids with one or more other lipids, membrane stabilizers or antioxidants not listed above.

[0069] The molar percentage of lipid in the bilayer membrane may be equal to or less than about 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or any value or range therebetween (e.g., about 5 to 50%, about 5 to 45%, about 5 to 40%, about 5 to 35%, about 5 to 30%, about 5 to 25%, about 5 to 20%, about 5 to 15%, or about 5 to 10%).

[0070] The lipid of the bilayer membrane can be a mixture of a first phospholipid and a second phospholipid. The first phospholipid can be selected from the group consisting of PC, HSPC, DOPC, POPC, DSPC, DPPC, DMPC, PSPC and combinations thereof, and the second phospholipid is selected from the group consisting of PE, PG, DOPE, PEG-DSPE, DPPG, DOPG, DOTAP, DOTMA, DDAB and combinations thereof. In other embodiments, the molar percentage of the first phospholipid in the bilayer membrane is about 50, 45, 40, 35, 30, 25, 20, 15, 10, or any number or range of values ​​therebetween (e.g., about 5-50%, about 5-45%, about 5-40%, about 5-35%, about 5-30%, about 5-25%, about 5-20%, about 5-15%, or about 5-10%), and the molar percentage of the second phospholipid in the bilayer membrane is between 0.1 and about 15, 14, 13, 12, 11, 10, 9, 8, 7, or any number or range of values ​​therebetween (e.g., about 0.1-15%, about 0.1-10%, about 0.5-15%, about 0.5-10%, or about 0.5-7%). In an exemplary embodiment, the molar ratio of the first phospholipid (DSPC) to the second phospholipid (DOPE, DOPG or DDAB) may be 4:1 to 6:1.

[0071] In an exemplary embodiment, the bilayer membrane of the liposome includes less than about 55 molar percent of a steroid, preferably cholesterol. The molar percent of the steroid (e.g., cholesterol) in the bilayer membrane may be about 15-55%, about 20-55%, about 25-55%, about 15-50%, about 20-50%, about 25-50%, about 15-45%, about 20-45%, about 25-45%, about 15-40%, about 20-40%, or about 25-40%. The molar percent of phospholipids to cholesterol in the bilayer membrane may be about 25-50%:15-55%, 25-50%:20-55%, or 25-50%:15-50%. The molar ratio of phospholipids to cholesterol may be 1:1 to 3:1. The molar percentages of the first phospholipid, the second phospholipid and cholesterol in the bilayer membrane may be about 25-50%:0.1-15%:15-55%, 5-50%:0.1-15%:10-40% or 25-50%:0.5-10%:5-20%.

[0072] Liposomes encapsulating the capture agent can be prepared by any technique currently known or subsequently developed. For example, MLV liposomes can be formed directly from a hydrated lipid film, spray-dried powder or freeze-dried cake of a selected lipid composition and a capture agent; SUV liposomes and LUV liposomes can be sized from MLV liposomes by ultrasonication, homogenization, microfluidization or extrusion.

[0073] In yet another embodiment, the compound comprising retinoic acid conjugated to a carbohydrate or a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition and the second pharmaceutical composition can be used to inhibit viral infection or replication. The virus may be an RNA virus. The RNA virus may include a coronavirus, a human immunodeficiency virus (HIV), a hepatitis C virus (HCV), an influenza virus, or any combination thereof. The coronavirus may include severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU (HCoV-HKU1), or any combination thereof. In an exemplary embodiment, the coronavirus is SARS-CoV-2 that causes COVID-19.

[0074] In yet another embodiment, the compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and the first pharmaceutical composition and the second pharmaceutical composition can be used to treat cancer. The cancer may include leukemia, lymphoma, carcinoma or sarcoma. In exemplary embodiments, the cancer may be lung cancer, ovarian cancer, breast cancer, liver cancer, pancreatic cancer or bile duct cancer.

[0075] In certain embodiments, the compound and the metal ion in the first pharmaceutical composition can be administered separately, simultaneously or sequentially. The retinoic acid and the carbohydrate in the second pharmaceutical composition can be administered separately, simultaneously or sequentially. All or at least two of the retinoic acid, carbohydrate and metal ion in the second pharmaceutical composition can be administered separately, simultaneously or sequentially. In an exemplary embodiment, the metal ion and the carbohydrate are administered simultaneously, followed by the retinoic acid.

[0076] In exemplary embodiments, the time interval between sequential administrations may be 1 to 30 minutes, 30 to 60 minutes, 60 to 90 minutes, 90 to 120 minutes, 2 to 3 hours, 3 to 12 hours, or 12 to 24 hours.

[0077] In certain embodiments, the compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and each of the first pharmaceutical composition and the second pharmaceutical composition can be prepared in dimethyl sulfoxide (DMSO), ethanol, a buffer or water for administration. The dosage and frequency of administration can vary according to the following factors: the severity of the viral infection (e.g., coronavirus infection) or the disease to be treated (e.g., cancer) and the weight, age, physical condition and response of the subject to be treated. The daily dose of the above therapeutic agent can be administered in a single dose or several doses.

[0078] In certain embodiments, the compound comprising retinoic acid conjugated to a carbohydrate, a pharmaceutically acceptable salt thereof, and the first and second pharmaceutical compositions are each administered orally, intravenously, intramuscularly, subcutaneously, intraperitoneally, intranasally, or topically.

[0079] Examples

[0080] The present invention will be further described by the following examples. However, it should be understood that the following examples are for illustrative purposes only and should not be construed as limiting the present invention in practice.

[0081] Materials and Methods

[0082] A. A novel retinoic acid compound represented by formula (Ia), i.e., galactose-modified isoretinoic acid, was prepared according to the following synthetic flow chart:

[0083]

[0084] I. General Experimental Methods

[0085] All reagents were commercially available and used without further purification. Yields refer to purified compounds identified as high purity by spectroscopy. Merck TLC Aluminum Flake Silica Gel 60F was used. 254 Plates were subjected to thin layer chromatography (TLC) and visualized by fluorescence quenching under UV light. Flash chromatography was performed using silica gel (Chromatorex, MB70-40 / 75, 40 to 75 μm) purchased from Fuji Silysia Chemicals. NMR spectra were recorded on a Varian-400MR operating at 400 MHz. 1 H. Chemical shifts are reported in ppm, with the solvent resonance as internal standard. Data are reported as follows: s = singlet, br = broad, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet; coupling constants are in Hz; integration was performed. Purity was recorded on a Waters e2695 Separation Module / 2998 PDA Detector HPLC system (column: XBridge C18, 5 μm, 4.6 mm (ID) x 150 mm (L), eluent: a mixture of mobile phases A and B, mobile phase A: 100% acetonitrile; mobile phase B: pure water containing 0.1% formic acid and 10 mM NH4OAc, flow rate: 0.5 mL / min. Detection: UV, 254 nm).

[0086] II. Synthesis of the compound (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(3-bromopropoxy)tetrahydro-2H-pyran-3,4,5-triyl triacetate (2)

[0087]

[0088] Boron trifluoride-diethyl etherate (8.04 mL, 64.0 mmol) was added to a solution of galactose pentaacetate (compound (1), 5.0 g, 13 mmol), 3-bromo-1-propanol (1.73 mL, 19.0 mmol) and freshly dried molecular sieves in anhydrous dichloromethane (50 mL) at 0°C. The resulting mixture was stirred at room temperature overnight. The solution was neutralized with triethylamine, the molecular sieves were removed by passing through celite, and the reaction mixture was washed with water and brine. The organic layer was dried over anhydrous magnesium sulfate and evaporated to dryness. The crude product was purified by silica gel column chromatography (EtOAc: hexane = 1:2) to obtain the desired product (2) (0.625 g, 10%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 5.40 (dd, J = 3.4, 1.2 Hz, 1H), 5.22-5.16 (m, 1H), 5.04-5.01 (m, 1H), 4.48 (d, J = 8 Hz, 1H), 4.21-4.11 (m, 2H), 4.03-3.98 (m, 1H), 3.92 (td, J = 5.8, 0.8 Hz, 1H), 3.72-3.66 (m, 1H), 3.50-3.47 (m, 2H), 2.25-1.99 (m, 14H); LCMS (ESI) m / z calculated for C 17 H 25 B O 10 469.28; found 491.04 [M+Na] + .

[0089] III. Synthesis of the compound (2R,3S,4S,5R,6R)-2-(acetoxymethyl)-6-(3-azidopropoxy)tetrahydro-2H-pyran 3,4,5-triyl triacetate (3)

[0090]

[0091] Sodium azide (0.43 g, 6.6 mmol) was added to a solution of DMF (4 mL) containing compound (2) (0.62 g, 1.3 mmol). The resulting mixture was stirred at 100° C. for 2 hours. The solution was concentrated to dryness. The crude product was purified by silica gel column chromatography (EtOAc: hexane = 1: 2) to obtain the desired product (3) (458 mg, 80%) as a colorless oil. 1 H NMR (400 MHz, CDCl3): δ 5.39 (dd, J = 3.4, 0.8 Hz, 1H), 5.22-5.18 (m, 1H), 5.03-5.00 (m, 1H), 4.48 (d, J = 8 Hz, 1H), 4.21-4.10 (m, 2H), 3.99-3.89 (m, 1H), 3.63-3.58 (m, 1H), 3.39-3.35 (m, 2H), 2.15 (s, 3H), 2.07 (s, 3H), 2.05 (s, 3H), 1.99 (s, 3H), 1.92-1.77 (m, 2H); LCMS (ESI) m / z calculated for C 17 H 25 N3O 10 431.40; found 454.2 [M+Na] + .

[0092] IV. Synthesis of the compound (2R,3R,4S,5R,6R)-2-(3-azidopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (4)

[0093]

[0094] Sodium methoxide (28 mg, 0.5 mmol) was added to a solution of dichloromethane (1.0 mL) and methanol (4.0 mL) containing compound (3) (0.45 g, 1 mmol). The resulting mixture was stirred at room temperature for 10 hours. The solution was neutralized with Dowex 50WX8 and evaporated to dryness to give the desired product (4), which was used in the next step without further purification. LCMS (ESI) m / z calculated as C9H 17 N3O6 263.25; found 286.1 [M+Na] + .

[0095] V. Synthesis of the compound (2R,3R,4S,5R,6R)-2-(3-aminopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (5)

[0096]

[0097] Crude compound (4) in methanol (10.5 mL) was treated with Pd / C (11 mg) and acetic acid (6.3 mg) under H2. The solution was filtered through a pad of celite and the filtrate was concentrated under reduced pressure to give (2R,3R,4S,5R,6R)-2-(3-aminopropoxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (5), which was used in the next step without further purification. LCMS (ESI) m / z calculated for C9H 19 NO6 237.25; found to be 237.8 [M] + .

[0098] VI. Synthesis of the compound perfluorophenyl (2Z, 4E, 6E, 8E) -3, 7-dimethyl-9- (2, 6, 6-trimethylcyclohex-1-en-1-yl) nona-2, 4, 6, 8-tetraenoate (6)

[0099]

[0100] Pentafluorophenyl trifluoroacetate (compound (8), 0.081 mL, 0.47 mmol) was added to a solution of anhydrous DMF (1.0 mL) containing retinoic acid (compound (7), 0.10 g, 0.33 mmol) and triethylamine (0.093 mL, 0.67 mmol) at 0°C. The solution was stirred at room temperature for 2 hours. The reaction mixture was diluted with EtOAc and washed with 0.1N aqueous HCl, followed by aqueous NaHCO3 and brine. The organic layer was dried over anhydrous magnesium sulfate and concentrated to dryness to obtain the desired product (6) without further purification.

[0101] VII. Synthesis of the target compound (2Z,4E,6E,8E)-3,7-dimethyl-N-(3-(((2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)propyl)-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenamide) (Ia)

[0102]

[0103] Triethylamine (0.049 mL, 0.33 mmol) was added to a solution of crude compound (5) (30 mg, 0.13 mmol) and active ester (6) (150 mg, 0.33 mmol) in DMF (1.0 mL) at room temperature. The resulting mixture was stirred overnight. The solution was concentrated to dryness. The crude product was purified by silica gel column chromatography (methanol: dichloromethane = 1:9). LCMS (ESI) m / z calculated as C 29 H 45 NO7 519.68; found 520.5 [M+H] + See also Figure 1 .

[0104] B. Preparation of SARS-CoV-2 PLpro

[0105] In order to determine whether the claimed compounds and pharmaceutical compositions can inhibit SARS-CoV-2, their in vitro inhibitory effects on the activity of SARS-CoV-2 papain-like protease (PLpro) were first evaluated.

[0106] The codon-optimized gene sequence encoding the wild-type SARS-CoV-2 PLpro was synthesized by Biotools Co., Ltd. and subcloned into the pET-21a (Novagen) vector using the NdeI and XhoI restriction enzyme sites, while the His tag coding region (-LEHHHHHH-) was retained at the C-terminus.

[0107] The vector into which the SARS-CoV-2 PLpro gene was inserted was transformed into the Escherichia coli BL21 (DE3) strain (Yeastern Biotech Co., Ltd.) to express PLpro in large quantities. The culture was carried out in LB medium (containing 1% tryptone, 0.5% yeast extract, and 1% NaCl) supplemented with ampicillin (100 μg / mL) as an antibiotic marker. The resulting culture was initially cultured at 37°C while shaking at 200 rpm. At 600 nm (OD 600) at an optical density between 0.6 and 0.8, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.4 mM to induce the expression of PLpro. The culture was continued at 18°C ​​and 200 rpm for 20 hours. The cells were harvested by centrifugation (5,000 x g) and disrupted by ultrasonic oscillation in a lysis buffer containing 50 mM sodium phosphate (pH 7.4), 1.0 mM DTT, 5% glycerol and 100 mM NaCl. Subsequently, the cells were centrifuged at 20,000 x g for 50 minutes to remove cell debris. The supernatant was loaded onto a 5 mL His-Trap HP column (GE Healthcare LifeSciences) and the protein therein was eluted using a gradient of 50 mM sodium phosphate (pH 7.4) and 100 mM NaCl containing 0 to 500 mM imidazole. The fractions containing His-tagged SARS-CoV-2 PLpro were pooled and concentrated using a Centricon membrane (10K cutoff, GE Healthcare Life Sciences). The His-tagged SARS-CoV-2 PLpro was further purified by gel filtration chromatography using a Superdex 75 gel filtration column (GE Healthcare Life Sciences) in 50 mM sodium phosphate buffer (pH 7.4). The concentration of PLpro was measured at 280 nm (using 45270 M -1 cm -1 The concentration of SARS-CoV-2 PLpro was determined by the ultraviolet absorbance based on the extinction coefficient (ε280).

[0108] C. SARS-CoV-2 PLpro activity test

[0109] The enzymatic activity of the SARS-CoV-2 PLpro obtained above was measured by a colorimetry-based peptide cleavage assay using a 6-mer peptide substrate, FRLKGG-p-nitroaniline (FG6-pNA) (HPLC purity 97%; GL Biochem Ltd., Shanghai, China). In the cleavage assay, the 6-mer peptide substrate is cleaved at the Gly-pNA bond to release free pNA, which turns the solution color yellow. The cleavage assay was performed using a 96-well microplate spectrophotometer (Epoch Biochem Ltd., Shanghai, China). TM 2, Biotek), continuously monitoring 405nm (A 405 ) to determine the enzyme activity.

[0110] Specifically, the cleavage test was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4), and FG6-pNA was added to each well to prepare a substrate solution having various FG6-pNA concentrations (0.1875 mM, 0.375 mM, 0.75 mM, 1.5 mM, 3.0 mM, 6.0 mM). The test mixture (180 μL per well) was pre-incubated for 10 minutes to precisely control the temperature, and the reaction was initiated by adding 20 μL of SARS-CoV-2 PLpro solution (1.75 μM) to the test mixture. The SARS-CoV-2 PLpro solution was prepared by mixing the SARS-CoV-2 PLpro obtained above with 50 mM sodium phosphate buffer (pH 7.4). By measuring A 405 , and use 9800M -1 cm -1 The extinction coefficient (ε405) (at 1mM, A 405 =9.8) to calculate the concentration of pNA released by proteolysis.

[0111] OriginPro 8.0 software (OriginLab Corporation, USA) was used to obtain steady-state enzyme kinetic parameters by fitting the initial velocity (V0) data based on the Michaelis-Menten equation. All measurements were performed in triplicate. The data obtained are expressed as mean ± standard deviation.

[0112] Result: K M With k cat The values ​​were 2.50±0.03mM and 0.85±0.01s -1 . Therefore, it was verified that according to the above-mentioned manufacturing method, SARS-CoV-2PLpro with protease activity was successfully prepared and can be used for subsequent SARS-CoV-2PLpro inhibition tests.

[0113] D. Establishment of SARS-CoV-2 animal model

[0114] Golden Syrian hamsters (5-6 weeks old, average weight of about 100 g) were obtained from the Experimental Animal Center. The hamsters were housed in an animal room with specific-pathogen-free (SPF) conditions commonly used in the art. In addition, all hamsters were provided with water and feed ad libitum. All experiments involving hamsters were reviewed and approved by the relevant institutions.

[0115] Hamsters were inoculated intranasally with 1 × 10 4Plaque forming units (PFU) were obtained at 12:00 PM and infected with phosphate buffered saline (PBS) containing SARS-CoV-2 (obtained from the P3 laboratory of the relevant institution; wild type) to establish a SARS-CoV-2 animal model. The established SARS-CoV-2 animal model was confirmed (data not shown).

[0116] Example 1

[0117] SARS-CoV-2 PLpro inhibition assay for in vitro evaluation of the antiviral effect of a novel retinoic acid compound (galactose-modified isoretinoic acid) against SARS-CoV-2

[0118] The enzyme inhibition test was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4). The SARS-CoV-2 PLpro (0.9 μM) obtained above was added to each well to form an enzyme solution. The enzyme solution in each well was divided into a control group (referred to as "control"), a comparison group (referred to as "comparative example 1") and an experimental group (referred to as "experimental example 1"). The corresponding inhibitors shown in Table 1 below were added to each group of enzyme solutions to form a test mixture (total volume of 180 μL). Pre-culture was performed for 30 minutes.

[0119] Table 1

[0120] Group Components Comparison PLpro Comparative Example 1 PLpro+50μM isoretinic acid Experimental Example 1 PLpro+50μM galactose-modified isoretinoic acid

[0121] 20 μL of the above FG6-pNA (1.2 mM) was added to each test mixture to initiate the enzyme reaction. The enzyme reaction was allowed to proceed at 30°C for 300 seconds. The 96-well microplate spectrophotometer (Epoch TM 2, Biotek), continuously monitoring 405nm (A 405 ) to determine the enzyme activity. The reaction rate is calculated based on this, where the reaction rate is the absorbance A 405 Slope versus time (seconds), total reaction time is 300 seconds. Data are presented in Figure 2 middle.

[0122] The percentage inhibition was calculated using the following formula:

[0123] A=[1-(B / C)]×100 (I)

[0124] Where A = percent inhibition

[0125] B = reaction rate of each group

[0126] C = reaction rate of the control group

[0127] All the data obtained were expressed as mean ± standard deviation.

[0128] Results: The inhibition percentages of different treatment groups are shown in Table 2 below.

[0129] Table 2

[0130]

[0131] like Figure 2 As shown in Table 2, the inhibition rate of Experimental Example 1 is significantly higher than that of Comparative Example 1, which indicates that galactose-modified isoretinoic acid is more effective than isoretinoic acid alone in inhibiting the activity of PLpro enzyme. This result shows that galactose-modified isoretinoic acid has a better inhibitory effect on the activity of papain-like protease.

[0132] Furthermore, it provides important insights into the biochemical properties of the PLpro family of coronaviruses and paves the way for promising therapeutic strategies against SARS-CoV-2.

[0133] Example 2

[0134] In vivo evaluation of the antiviral effect of a novel retinoic acid compound (galactose-modified isoretinoic acid) against SARS-CoV-2 in an animal model of SARS-CoV-2

[0135] Since the novel retinoic acid compound galactose-modified isoretinoic acid has been shown to have an inhibitory effect on SARS-CoV-2 in vitro, the antiviral effect of the novel retinoic acid compound on SARS-CoV-2 was further evaluated in in vivo animal tests.

[0136] The infected hamsters obtained above were divided into the following four groups (n=5 in each group): a control group (referred to as "control"), two comparative groups (referred to as "comparative example 1" and "comparative example 2") and an experimental group (referred to as "experimental example 1"). The therapeutic agents used in each group are listed in Table 3 below.

[0137] Table 3

[0138]

[0139] Specifically, for each hamster in Experimental Example 1, the novel retinoic acid compound was intranasally administered at a dose of 0.35 mg / kg at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, Day 1) and at a dose of 0.35 mg / kg at 8:00 PM on the day of infection. In the next two days (Day 2 and Day 3), the novel retinoic acid compound was administered nasally twice a day at 8:00 AM and 8:00 PM at a dose of 0.35 mg / kg each.

[0140] For each hamster in the comparison group, the isoretinic acid in Comparative Example 1 or the liposome-encapsulated isoretinic acid in Comparative Example 2 was intranasally administered at 0.35 mg / kg at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection and at 0.35 mg / kg at 8:00 PM on the day of infection. In the next two days after the infection day, the isoretinic acid in Comparative Example 1 or the liposome-encapsulated isoretinic acid in Comparative Example 2 was intranasally administered at a dose of 0.35 mg / kg twice a day at 8:00 AM and 8:00 PM. The liposome-encapsulated isoretinic acid in Comparative Example 2 was prepared by a related agency using commonly used techniques in the art and is described in detail above. Since the main technical feature of the present invention is a novel retinoic acid compound, i.e., galactose-modified isoretinic acid, the details of the liposomes are omitted here for the sake of brevity.

[0141] For each hamster in the control group, buffer was administered nasally at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection at 100 μL and at 8:00 PM on the day of infection at 100 μL. For two days after the infection day, buffer was administered nasally at 8:00 AM and 8:00 PM twice a day at 100 μL.

[0142] After 3 days of treatment, the hamsters were sacrificed and their lungs were collected for analysis by TCID 50 The assay measures the live viral load in Vero E6 cells. The 50% tissue culture infectious dose (TCID 50 ) was used to determine the virus titer. All experiments on SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by relevant agencies.

[0143] Experimental data were analyzed using Tukey's test to assess differences between groups. Statistical significance was indicated at p < 0.05.

[0144] Results: See Figure 3 , TCID of Experimental Example 1 50 The TCID of Comparative Example 1 and Comparative Example 2 was significantly higher than that of 50 This indicates that the novel retinoic acid compound, i.e., galactose-modified isoretinoic acid, has higher in vivo efficacy against SARS-CoV-2 than isoretinoic acid alone or liposome-encapsulated isoretinoic acid alone.

[0145] Example 3

[0146] Evaluation of the in vitro cytotoxic effects of a novel retinoic acid compound (galactose-modified isoretinoic acid) on different cancer cell lines

[0147] In this example, seven cell lines were used, namely AsPC-1, MDA-MB-231, HCT-116, Huh-7, SKOV-3, A549 and H460 cancer cell lines. Each cell line was cultured in a designated growth medium containing 10% FBS and in a 37°C humidified incubator containing 5% CO2.

[0148] The day before treatment, cells in the logarithmic growth phase were collected, counted, and analyzed by 1×10 4 The cells were seeded at a density of 10 cells / 100 μL / well in a 96-well plate. After overnight culture, the medium in each well was gently removed, and then fresh medium (200 μL / well) was added.

[0149] The test article (TA) is a novel retinoic acid compound (referred to as "novel compound" in this example), which is freshly prepared as a 96mM stock solution in 100% DMSO on the day of treatment. Isoretine acid is serially diluted 2-fold using 100% DMSO to obtain different concentrations of isoretine acid from 7.5mM to 96mM. Cells are treated by adding 2.02μL of the specified concentration of isoretine acid to each well to obtain a final concentration range of 7.5μM to 960μM of isoretine acid, and all wells are maintained with 1% DMSO, including DMSO controls. All components are gently mixed and cultured for 24 hours.

[0150] Cells were treated by adding 2.02 μL of the indicated concentration of novel Compound A or DMSO to each well, followed by gentle mixing and incubation for 24 hours.

[0151] On the day of measuring cell viability, the medium of each well was replaced with freshly prepared Alamar blue dye (10% v / v) and incubated for 2 to 3 hours at 37° C. Spectroscopic absorbance was recorded at wavelengths of 570 nm and 600 nm.

[0152] The percentage of cell viability was calculated using the following formula:

[0153] % survival rate = (TA [A 570 / A 600 ratio] / DMSO [A 570 / A 600 Ratio])×100%

[0154] Where TA = object to be tested

[0155] A 570 = absorbance at 570 nm

[0156] A 600 = absorbance at 600nm

[0157] Results: See Figures 4 to 10 , which shows that the novel compound can significantly reduce the cell survival rate of all seven cancer cell lines, which shows that the novel retinoic acid compound, i.e., galactose-modified isoretinoic acid, can be a potential anticancer drug for the effective treatment of various cancers (such as lung cancer, ovarian cancer, breast cancer, pancreatic cancer, colorectal cancer and liver cancer). Table 4 shows IC 50 .

[0158] Table 4

[0159] Tumor type Cell lines <![CDATA[IC 50 (μM)]]> Non-small cell lung cancer NCI-H460 22.95 Non-small cell lung cancer A549 21.88 Liver cancer Huh-7 77.46 Pancreatic cancer AsPC-1 23.00 Colorectal cancer HCT-116 123.44 Ovarian cancer SKOV-3 13.82 Breast cancer MDA-MB-231 115.88

[0160] Example 4

[0161] SARS-CoV-2 PLpro inhibition assay for in vitro evaluation of the antiviral effect of a second pharmaceutical composition on SARS-CoV-2

[0162] The enzyme inhibition test was performed in a 96-well microplate. Each well of the microplate contained 50 mM phosphate buffer (pH 7.4). The SARS-CoV-2 PLpro obtained above was added to each well to form an enzyme solution with a final concentration of 0.9 μM. The enzyme solution in each well was divided into a control group (referred to as "control"), four comparison groups (referred to as "Comparative Example 1", "Comparative Example 2", "Comparative Example 3" and "Comparative Example 4", respectively) and two experimental groups (referred to as "Experimental Example 1" and "Experimental Example 2", respectively). The corresponding inhibitors shown in Table 5 below were added to each group of enzyme solutions to form a test mixture (total volume of 180 μL). Pre-culture was performed for 30 minutes.

[0163] Table 5

[0164]

[0165] 20 μL of the above FG6-pNA (1.2 mM) was added to each test mixture to initiate the enzyme reaction. The enzyme reaction was allowed to proceed at 30°C for 300 seconds. The 96-well microplate spectrophotometer (Epoch TM 2, Biotek), continuously monitoring 405nm (A 405 ) to determine the enzyme activity. Based on this, the reaction rate is calculated, where the reaction rate is the absorbance A 405 Slope versus time (seconds), total reaction time is 300 seconds. Data are presented in Fig.11 The percentage inhibition was calculated as described in Example 3.

[0166] Results: The inhibition percentages of different treatment groups are shown in Table 6 below.

[0167] Table 6

[0168]

[0169] like Fig.11 As shown in Table 6, the inhibitory effects of the experimental groups were significantly higher than those of the comparative groups, which indicated that the two experimental groups: (1) isoretinic acid and oligosaccharides, and (2) isoretinic acid, oligosaccharides, Zn 2+ Mg 2+ , compared with only isoretinic acid, oligosaccharides, and Zn 2+ or Mg 2+ The individual components are more effective. This result shows that isoretinic acid, oligosaccharides and divalent metal ions have a synergistic inhibitory effect on papain-like protease activity. In addition, it provides important insights into the biochemical properties of the coronavirus PLpro family and leads the way to promising therapeutic strategies against SARS-CoV-2.

[0170] Example 5

[0171] Evaluation of the in vivo therapeutic effects of isoretinic acid, oligosaccharides or divalent metal ions on SARS-CoV-2 in animal models

[0172] The infected hamsters obtained in Part A of this experiment were divided into the following six groups (n=5 in each group): one control group, three experimental groups (i.e., Experimental Example 1, Experimental Example 2, and Experimental Example 3), and four comparative groups (i.e., Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4). The therapeutic agents used in each group are listed in Table 7 below.

[0173] Table 7

[0174]

[0175] The liposome-encapsulated therapeutic agent is prepared by the relevant institutions using the commonly used technology in the field as liposomes and is described in detail above. Since the main technical feature of the present invention is the combination of retinoic acid and oligosaccharides with or without metal ions, the details of the liposomes are omitted here for the sake of brevity.

[0176] Specifically, for each hamster in the experimental group, liposome-encapsulated isoretinoic acid was intranasally administered at 0.35 mg / kg at 8:00 AM on the day of infection (i.e., 4 hours before SARS-CoV-2 infection, day 1) and at 0.35 mg / kg at 8:00 PM on the day of infection. In the next two days (days 2 and 3), liposome-encapsulated isoretinoic acid was administered intranasally at 0.35 mg / kg twice a day at 8:00 AM and 8:00 PM.

[0177] In addition to intranasal administration of 0.35 mg / kg of liposome-encapsulated isoretinoic acid twice a day at 8:00 AM and 8:00 PM, 15 μL of liposome-encapsulated oligosaccharide mixture (i.e., Experimental Example 1) or oligosaccharide mixture (15 μL) and Zn were intranasally administered once a day from 7:30 PM to 8:00 PM on the day of infection (i.e., 0.5 to 1 hour before the administration of liposome-encapsulated isoretinoic acid in the evening). 2+ (100 μM), Mg 2+ (200μM) and K + (200 μM)(15 μL), both of which were encapsulated in liposomes with a total volume of 30 μL (ie, Experimental Example_2), and in the next two days, 15 μL of Experimental Example_1 or 30 μL of Experimental Example_2 was administered.

[0178] For each hamster in the comparison group, the isoretinoic acid in Comparative Example 1 or the liposome-encapsulated isoretinoic acid in Comparative Example 2 was intranasally administered at 0.35 mg / kg at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection and at 0.35 mg / kg at 8:00 PM on the day of infection. In the next two days after the infection day, the isoretinoic acid in Comparative Example 1 or the liposome-encapsulated isoretinoic acid in Comparative Example 2 was intranasally administered at 0.35 mg / kg twice a day at 8:00 AM and 8:00 PM.

[0179] For each hamster, the liposome-encapsulated Zn prepared in Comparative Example 3 was administered once a day through the nasal cavity from 7:30 PM to 8:00 PM on the day of infection. 2+ (100 μM), Mg 2+ (200μM) and K + (200 μM) and the liposome-encapsulated oligosaccharide mixture of Comparative Example 4 were administered in a volume of 15 μL each on the next two days.

[0180] For each hamster in the control group, buffer was administered nasally at 8:00 AM (i.e., 4 hours before SARS-CoV-2 infection) on the day of infection at 100 μL and at 8:00 PM on the day of infection at 100 μL. For two days after the infection day, buffer was administered nasally at 8:00 AM and 8:00 PM twice a day at 100 μL.

[0181] After 3 days of treatment, the hamsters were sacrificed and their lungs were collected for analysis by TCID 50 The assay measures the live viral load in Vero E6 cells. The 50% tissue culture infectious dose (TCID 50) was used to determine the virus titer. All experiments on SARS-CoV-2 were performed in a biosafety level 3 (BSL-3) laboratory and approved by relevant institutions.

[0182] Experimental data were analyzed using Tukey's test to assess differences between groups. Statistical significance was indicated at p < 0.05.

[0183] Results: See Fig.12 , TCID of each of Experimental Example 1 and Experimental Example 2 50 The TCID of each of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4 is significantly higher than that of each of Comparative Example 1, Comparative Example 2, Comparative Example 3 and Comparative Example 4. 50 This indicates that the combination of isoretinoic acid and oligosaccharides with / without metal ions has higher in vivo efficacy against SARS-CoV-2 compared with isoretinoic acid alone, metal ions alone, or oligosaccharides alone.

[0184] in conclusion

[0185] In view of the results of Examples 1 to 5, it is verified that the claimed novel retinoic acid compound or its pharmaceutically acceptable salt, or the claimed pharmaceutical composition comprising the compound (i.e., the first pharmaceutical composition), or the claimed pharmaceutical composition comprising retinoic acid and carbohydrates with / without metal ions (i.e., the second pharmaceutical composition) can provide improved and / or synergistic effects in inhibiting the infection and replication of SARS-CoV-2 and treating diseases associated with SARS-CoV-2 infection or cancer. Therefore, the compound comprising retinoic acid conjugated to carbohydrates, its pharmaceutically acceptable salt, and the first pharmaceutical composition and the second pharmaceutical composition described in the present invention can indeed be used as drug-repurposing agents.

[0186] The invention has been described broadly and throughout this document. Each narrower form and subgroup falling within the overall disclosure also forms part of the invention. In addition, where features or aspects disclosed herein are described in terms of Markush groups, one of ordinary skill in the art will appreciate that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the compound comprises retinoic acid conjugated to a carbohydrate.

2. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the compound is represented by formula (I): Where R 1 is a substituted or unsubstituted functional group of the carbohydrate.

3. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the carbohydrate is selected from the group consisting of monosaccharides, disaccharides, oligosaccharides and polysaccharides.

4. The compound or pharmaceutically acceptable salt thereof according to claim 3, wherein the oligosaccharide is a mono-oligosaccharide or a hetero-oligosaccharide; and the polysaccharide is a mono-polysaccharide or a hetero-polysaccharide.

5. The compound of claim 3 or a pharmaceutically acceptable salt thereof, wherein the carbohydrate comprises glucose, fructose, galactose, mannose, sucrose, lactose, maltose, β-1,3 / 1,6-glucan oligosaccharides, raffinose, stachyose, verbascose, fructooligosaccharides, starch, glycogen, cellulose, or any combination thereof. 6 . A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier comprises liposomes, and the compound or a pharmaceutically acceptable salt thereof is encapsulated by the liposomes. The pharmaceutical composition according to claim 6 , further comprising a metal ion.

9. The pharmaceutical composition of claim 8, wherein the metal ions comprise monovalent ions, divalent ions, or a combination thereof.

10. The pharmaceutical composition of claim 9, wherein the monovalent ions comprise K + 、Na + or a combination thereof; and the divalent ion comprises Zn 2+ Mg 2+ , Cu 2+ , Mn 2+ , Ca 2+ , Fe 2+ or any combination thereof.

11. The pharmaceutical composition according to claim 8, wherein the pharmaceutically acceptable carrier comprises liposomes, and the metal ions are encapsulated by the liposomes. 12 . The pharmaceutical composition according to claim 11 , wherein the compound or a pharmaceutically acceptable salt thereof and the metal ion are encapsulated by the liposome alone or simultaneously.

13. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 6 to 12, in the manufacture of a medicament for inhibiting viral infection or replication.

14. The use according to claim 13, wherein the virus is an RNA virus.

15. The use according to claim 14, wherein the RNA virus comprises a coronavirus, human immunodeficiency virus (HIV), hepatitis C virus (HCV), influenza virus or any combination thereof.

16. The use according to claim 15, wherein the coronavirus comprises severe acute respiratory syndrome coronavirus (SARS-CoV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Middle East respiratory syndrome coronavirus (MERS-CoV), human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), human coronavirus NL63 (HCoV-NL63), human coronavirus HKU (HCoV-HKU1) or any combination thereof.

17. Use of the compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 6 to 12, in the manufacture of a medicament for treating cancer.

18. The use according to claim 17, wherein the cancer comprises leukemia, lymphoma, carcinoma or sarcoma.

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